Project Summary: The goal of this proposal is to characterize a protein complex called the myddosome, which is uniquely assembled during Toll-like Receptor (TLR) signal transduction. The myddosome is one of several newly- defined receptor-proximal complexes that control innate immune signal transduction. Analogous complexes operate to control inflammasome assembly and the RIG-I and TNF receptor signaling pathways. A common feature of these complexes is that they operate as organelles that are assembled ?on-demand?, or upon ligand binding. As such, these complexes are only present in cells after microbes have been detected, and they may serve as critical hubs for coordinating downstream signaling enzyme activation. Despite the recognition that these organelles control innate immunity, we have little understanding of their regulation (or composition). Our proposal is founded on our recent discovery that the myddosome is assembled within macrophages upon treatment with TLR ligands. This finding is important because it had been unclear whether the myddosome is a pre-existing protein complex, or if it is assembled inducibly. The inducible assembly of this complex provided us with a tool to study its regulation, and we recently identified the TLR sorting adaptor TIRAP as the first regulator of myddosome assembly. These discoveries establish the myddosome as an important model to study receptor-proximal protein complexes that define the signaling pathways of the innate immune system. In this application, we propose to explore how known myddosome components interact to regulate TLR signaling in vitro and in vivo (Aim 1), to explore how a new myddosome component regulates TLR signaling (Aim 2), and to explore the mechanisms underlying several mutant myddosome components that cause mouse or human disease (Aim 3).